Abstract Yersinia pestis is the causative agent of plague and has caused widespread loss of human life during three pandemics. Y. pestis is also firmly established in animal reservoirs on nearly every inhabited continent, with the largest reservoir in the southwestern United States. Each year multiple outbreaks occur resulting in 1,000- 5,000 human cases reported worldwide (likely an underestimate). Currently there is not a FDA approved vaccine for plague and the bacterium has a high potential for bioterrorism misuse. Consequently, Y. pestis is considered a Tier 1 Select Agent. In order to grow, Y. pestis must acquire essential biometals such as Fe, Mn and Zn. However, the host restricts access to these metals through nutritional immunity. Therefore, Y. pestis, like other pathogens, has evolved high affinity metal acquisition systems that are essential for virulence. We recently discovered that the yersiniabactin (Ybt) synthetase system not only contributes to Fe acquisition, but also to Zn acquisition in Y. pestis. Furthermore, we have shown that the inner membrane permease YbtX (which is not required for Fe-Ybt acquisition) is required for Ybt synthetase-dependent Zn acquisition. These discoveries have opened a new area in Ybt and Zn acquisition research. Specifically, we hypothesize that Y. pestis produces a Ybt synthetase-dependent zincophore that is distinct from the Ybt siderophore. Furthermore, Y. pestis uses a dedicated uptake system to reacquire this zincophore once bound to Zn. Along with ZnuABC, this new system is essential for Zn acquisition during infection. In Specific Aim 1, we will use a biochemical approach to purify and define the chemical structure and Zn affinity of the secreted Ybt synthetase-dependent zincophore. In Specific Aim 2, we will determine the contribution of the Ybt synthetase-dependent zincophore to virulence using a hemochromatosis mouse model. Importantly, the hemochromatosis mouse is defective in Fe nutritional immunity. Thus, this model will allow us to separate contributions of Ybt synthetase-dependent Fe acquisition from Ybt synthetase-dependent Zn acquisition to Y. pestis virulence. Finally, as Y. pestis also infects fleas for transmission, we will determine the contribution of Zn acquisition to colonization and survival in the flea vector. Completion of these aims will provide a comprehensive understanding of the role of Zn acquisition in Y. pestis virulence. Furthermore, as Ybt is a conserved virulence factor, this work will also be relevant to other bacterial pathogens that use the Ybt system.